This invention relates to methods of, and apparatus for, preparing a blood smeared slide for analysis and more particularly to spinning the slide for a time which is a function of the red cell concentration of the blood.
In the analysis of blood samples, the blood is smeared on a laboratory slide and the smear is stained. By counting the leukocytes on the stained smear, laboratory technicians perform what is refered to as a white blood cell differential. Automation of this differential has significant enconomic impact because the differential is performed so frequently at every hospital. A thesis by J. W. Bacus, "An Automated Classification of the Peripheral Blood Leukocytes by Means of Digital Image Processing", University of Illinois, Chicago, 1971, describes one automated system.
Copending application Ser. No. 353,004 filed Apr. 20, 1973, by Douglas A. Cotter, "Image Scanning Converter for Automated Slide Analyzer", now U.S. Pat. No. 3,883,852, describes a system developed by my co-workers for automatically scanning and determining the relative number of different types of leukocytes on the stained smear.
Centrifugally spinning a blood wetted slide to produce a monolayer blood film is described in a paper by M. Ingram and F. M. Minter, "Semi-automatic Preparation of Coverglass Blood Smears Using a Centrifugal Device," Amer. J. Clin. Path. 51: 214-221, 1969. The method described in this paper includes flooding a coverglass with a layer of blood and centrifuging the coverglass rapidly in a plane parallel to the plane of rotation of the centrifuge. Excess blood is spun off leaving a monolayer of well spread blood cells on the cover glass.
Centrifuges for spinning blood smeared slides are commercially available. Such devices are available from: Plate General Corporation, (sold by PEI, Inc. 947 Old York Rd., Abington, Pennsylvania); Perkin-Elmer Corp., 50 Danbury Rd., Wilton, Conn.; and Shandon Scientific Co. Inc., 515 Broad St., Sewickley, Pennsylvania.
While some commercially available blood spinning apparatus have controls for adjusting the spin time, it has been the practice to set this spin time to one position and to allow it to remain there for all blood slide preparations.
After use of the centrifuges and blood spinning techniques described above, we have made the following observations.
The separation of the red cells was not the same for all blood samples. For some bloods the spinning resulted in blood films with sparsely populated areas interspersed with clumps of cells. For other bloods the technique produced a slide with overlapping cells.
As mentioned in the article by Ingram, the morphology of the red cells was often altered. The cells appeared overly flattened and noncircular. Often, white blood cells (specifically neutrophils appeared damaged.
For the blood film to be uniform, a large quantity of blood had to be used. Typically, the surface was flooded prior to spinning. If the entire surface was not wetted an irregular "sunburst" pattern of the blood resulted.
Manual methods for obtaining a blood smear (wedge and cover-slide method) require a skilled operator, are not very reproducible, and produce distributions which are non-uniform, often containing a high percentage of damaged cells.
U.S. Pat. Nos. 3,577,267 Preston et al. and 3,705,048 Staunton describe centrifuges which can be used to prepare blood slides but the apparatus described in these patents does not solve the problem of producing blood smears with good cell morphology and good cell distribution for all blood samples.
Accordingly, we have concluded that the preparation of a slide which is suitable for an automated white cell differential is a critical task which must be performed by a machine operated by an operator who need not make subjective judgments in order to get reproducible results.